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A pair of modified coelenterazines has been created that enables convenient, sensitive real-time measurement of Renilla luciferase in living cells. One modified substrate generates very stable luminescence, allowing for the monitoring of dynamic cellular events in situ. The other substrate generates very intense luminescence in cells expressing Renilla luciferase, up to 3 or 4 times brighter than unmodified coelenterazine, and more than 10 times brighter than firefly luciferase with luciferin. These new modified coelenterazines were used to monitor Renilla luciferase activity in a CRE-responsive reporter-gene system, an RNAi-control system, and a BRET system (Bioluminescence Resonance Energy Transfer). For each system, conclusions derived from experiments using the new modified coelenterazines were consistent with those from the standard assay for that system, and the data was easier to generate because of the simple add-and-read procedure.

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